Mucin type O-glycosidically linked oligosaccharides have been described on a wide variety of protein molecules (Sadler, 1984). These structures constitute essential components in an equally wide variety of biological functions (e.g., Paulson, 1989; Jentoft, 1990 and references therein). The initial reaction in the biosynthesis of O-linked oligosaccharides is the transfer of N-acetylgalactosamine from the nucleotide sugar, UDP-N-acetylgalactosmine, to a serine or threonine residue on the acceptor polypeptide. This reaction, which can occur post-translationally, is catalyzed by a GalNAc-transferase enzyme (GalNAcT) called, UDP-GalNAc:polypeptide,N-acetylgalactosaminyltransferase. This is an intracellular membrane bound enzyme believed to be localized in the secretory pathway.
The exact location(s) of GalNAc-transferases in in vivo systems is not precisely known. It has been reported that the initial addition of N-acetylgalactosamine to the acceptor protein can take place early (even co-translationally) in the rough endoplasmic reticulum (ER). Other authors have suggested that this reaction is a post-translational event occurring in later ER compartments and/or in the cis region of the Golgi complex (e.g. Hanover et al. (1982) J. Biol. Chem. 257:10172-10177; Roth (1984) J. Cell Biol. 98:399-406; Elhammer and Kornfeld (1984) J. Cell Biol. 98:327-331; Tooze et al. (1988) J. Cell Biol. 106:1475-1487; Deschuyteneer et al. (1988) J. Biol. Chem. 263:2452-2459; Ulmer and Palade (1989) Proc. Natl. Acad. Sci. (U.S.A.) 89:663-667; Wertz et al. (1989) J. Virol. 63:4767-4776; Piller et al. (1989) Eur. J. Biochem. 183:123-135; Piller et al. (1990) J. Biol. Chem. 265:9264-9271.
Evidence has also been presented for a model in which transfer of N-acetylgalactosamine to Ser/Thr may occur in several compartments in the secretory pathway, including compartments later than the Golgi complex (Schachter and Brockhausen (1992) in Glycoconjugates, Allen and Kisailus, eds., pp. 263-332, Marcel Dekker Inc., New York). Elongation and termination of O-linked oligosaccharides is accomplished by sequential addition of individual monosaccharides by specific transferases (Roseman (1970) Chem. Phys. Lipids 5:270-280); current data suggest that these reactions are localized primarily in the Golgi apparatus (Schachter and Brockhausen, supra).
Enzyme-mediated synthesis of O-glycosidically linked oligosaccharides offer significant advantages over the classical synthetic organic pathways, producing very high yields of carbohydrates (e.g., oligosaccharides and/or polysaccharides), under mild conditions in aqueous solutions, and without generating notable amounts of undesired side products. However, an absolute prerequisite for this type of synthesis is the availability of cloned glycosyltransferases.
Endogenous enzymes can be isolated from most eucaryotic sources; however, these proteins are only found in low concentrations, so this is generally a difficult, time consuming procedure, yielding amounts of purified enzymes which are insufficient for in vitro synthesis work. Another complication is that the endogenous enzymes invariably are membrane bound, this complicates purification and in vitro uses of the enzyme. A cloned enzyme, on the other hand, can usually be expressed as a soluble enzyme with comparative ease.
In light of the considerable value of carbohydrates, there is accordingly a strong felt need for fast and quick assays of GalNAc-transferase.
Assays for GalNAc-transferase activity typically involve incubation of the activity containing preparation with radioactively labeled UDP-GalNAc and either an intact acceptor protein (e.g., basic myelin protein), a fragment(s) of a deglycosylated protein (e.g., various apomucins) or a synthetic peptide (e.g., Hagopian et al., 1971; Hagopian and Eylar, 1968; Young et al., 1979; Wang et al., 1992; Elhammer et al., 1993). The acceptor (sequence) requirements of the enzyme have recently been elucidated to a considerable extent and synthesis of efficient acceptor peptides can be accomplished quite readily (O'Connel et al., 1992; Wang et al., 1993; Elhammer et al., 1993). Following transfer of the radioactive sugar to the polypeptide acceptor, the product is isolated and the amount of enzymatic transfer quantitated by measuring the amount of radioactivity incorporated into the acceptor. Thus, in principle, assays for GalNAc-transferase activity are comparatively straight-forward.
A considerable technical problem associated with these assays, however, is the isolation of the glycosylated reaction product. For peptide acceptors, methods employed to date include chromatography on ion-exchange, size exclusion or reverse phase columns and, for protein acceptors, various (e.g., TCA) precipitation procedures. In the former case, a number of chromatography columns have to be prepared, equilibrated and developed for each experiment, in the latter, extensive washing procedures have to be carried out in order to reduce background radioactivity. Hence, typical GalNAc-transferase assays (using either acceptor) are both labor intense and time-consuming.
Here we describe a novel approach for the quantitation of the reaction products in GalNAc-transferase assays that employs fewer steps, takes less time, and is much more suitable than other assays currently available for screening for GalNAc-transferase.